Process for preparation of electrophoresis gel material

ABSTRACT

Describes a process for controlling the polymerization and cross-linked density of electrophoretic gel products useful for separation of bioorganic molecules, which process utilizes photoinitiation. Photoinitiated polymerized gels afford the desired advantages of being ultra thin and having a high electrophoretic resolution with programmable porosity profiles.

This is a division of application Ser. No. 625,840, filed June 28, 1984.

FIELD OF THE INVENTION

This invention relates to electrophoretic gel products of controlledcross-link density, useful for separation of bioorganic molecules, andto photopolymerization processes for their production.

BACKGROUND OF THE INVENTION

Electrophoresis is based on the principle that charged molecules orsubstances will migrate when placed in an electric field. Since proteinsand other biopolymers (e.g., DNA, RNA, enzymes and carbohydrates) arecharged, they migrate at pH values other than their isoelectric point.The rate of migration depends, among other things, upon the chargedensity of the protein or biopolymer and the restrictive properties ofthe electrophoretic matrix. The higher the ratio of charge to mass thefaster the molecule will migrate.

Many support media for electrophoresis are in current use. The mostpopular are sheets of paper or cellulose acetate, silica gels, agarose,starch, and polyacrylamide. Paper, cellulose acetate, and thin-layersilica materials are relatively inert and serve mainly for support andto minimize convection. Separation of proteins using these materials isbased largely upon the charge density of the proteins at the pH selectedand in general do not provide high resolution separations.

On the other hand, starch, agarose and polyacrylamide gel materials notonly minimize convection and diffusion but also actively participate inthe separation process. These materials provide a porous medium in whichthe pore size can be controlled to approximate the size of the proteinmolecules being separated. In this way, molecular sieving occurs andprovides separation on the basis of both charge density and molecularsize.

The extent of molecular sieving is thought to depend on how closely thegel pore size approximates the size of the migrating particle. The poresize of agarose gels is sufficiently large that molecular sieving ofmost protein molecules is minimal and separation is based mainly oncharge density. In contrast, polyacrylamide gels can have pores thatmore closely approximate the size of protein molecules and so contributeto the molecular sieving effect. Polyacrylamide has the furtheradvantage of being a synthetic polymer which can be prepared in highlypurified form.

The ability to produce a wide range of gel pore sizes and to form poresize gradients within the gel are additional advantages ofpolyacrylamide. Control over pore size enables mixtures to be sieved onthe basis of molecular size and enables molecular weight determinationsto be performed. These determinations are especially accurate if theproteins are coated with a detergent such as sodium dodecyl sulfate(SDS) which neutralizes the effects of molecular charge. This techniqueis referred to as SDS-PAGE electrophoresis. However techniques shown inthe prior art for preparing SDS Electrophoresis gels doe not hield ahighly reproducible product.

PORE GRADIENT GELS

Polyacrylamide gels can be made with a gradient of increasing acrylamideconcentration and hence decreasing pore size. These gels are now usedextensively instead of single concentration gels, both for analysis ofthe protein composition of samples and for molecular weight estimationusing SDS as a denaturing agent to render the proteins in a uniformcharge environment. The current techniques used to produce gradient gelsare both expensive and require great care to insure somewhatreproducible gradients.

Step gradients in which gels of different concentration are layered oneupon the other have been used. Unfortunately, they tend to giveartifactual multi-component bands at the interface between layers whichcreates inaccuracies in the protein mixture determination. It is nowcommon to use continuous acrylamide gradients. The usual limits are 3 to30% acrylamide in linear or nonlinear gradients with the particularrange chosen depending upon the size of the proteins to be fractionated.During electrophoresis in gradient gels, proteins migrate until thedecreasing pore size impedes further progress. Once this "pore limit" isapproached the protein banding pattern does not change appreciably withtime although migration does not cease completely.

One of the main advantages of gradient gel electrophoresis is that themigrating proteins are continually entering areas of gel with decreasingpore size such that the advancing edge of the migrating protein zone isretarded more than the trailing edge, resulting in a marked sharpeningof the protein bands. In addition, the gradient in pore size increasesthe range of molecular weights which can be fractionated simultaneouslyon one gel. Therefore a gradient gel will not only fractionate a complexprotein mixture into sharper bands than is usually possible with a gelof uniform pore size, but also can permit the molecular weightestimation of almost all the components.

Pore gradient gels are conventionally prepared by mixing high and lowconcentration monomer solutions in order to produce a concentrationgradient of acrylamide in the gel molds. These techniques incorporateelaborate pumping schemes which must be operated with great care toinsure the desired gradient. Also, these techniques are time consumingand expensive and do not lend themselves to reproducible productiontechniques.

In addition to the gradient in acrylamide concentration, a densitygradient of sucrose or glycerol is often included to minimize mixing byconvective disturbances caused by the heat of polymerization. Someworkers avoid the latter problem by including a gradient ofpolymerization catalyst to ensure that polymerization occurs first atthe top of the gel and then proceeds to the bottom. Gradient gelsprepared by any of these methods generally display poor reproducibility.

The polyacrylamide gel so prepared results from polymerization ofacrylamide and simultaneous polymer cross-linking by bifunctionalcompounds such as N,N'-methylene-bis-acrylamide (BIS). The mostprevalent method of initiation of polymerization is through the use ofsodium or ammonium persulfate an and acceleratorN,N,N',N'-tetramethylethylenediamine (TEMED). Acrylamide plymerizationcan also be accomplished photochemically using riboflavin. However,riboflavin initiated polymerization requires an intense UV or visiblelight source which must be applied for periods of 30 minutes to severalhours depending upon the intensity and wavelength of the source. Furtherdetrimental to its use is the fact that it undergoes a rapidphotobleaching reaction which reduces its concentration and changes itto a noninitiating form, so that achievement of high monomer conversionto polymer is difficult.

Photoinitiation with riboflavin is characteristically nonreproducibleboth because of the long duration of the exposure required withattendant uncertainty about complete polymerization and the chemicalinstability of riboflavin. Photoinitiation of polymerization asdescribed in the prior art does not allevaite the problems ofconstructing pore-gradient gels by mixing different concentrations ofmonomer liquids. In fact, the slow rate of polymerization withriboflavin and prolonged exposure with intense light sources only seemsto exaggerate the effects of convective mixing in destroying the designand reproducibility of the pore-gradient.

A. Chrambach and D. Rodbard (Sep. Sci. 7, 663-703, 1972) cited theacceleration of riboflavin photoinitiated polymerization by addition ofthe accelerator TEMED. For good control of the reaction, however thesystem also required persulfate be present. This system as withribloflavin alone does not alleviate the problems of constructingpore-gradient gels by mixing different concentrations of monomer. Inboth cases, riboflavin alone or with added accelerator (TEMED) andpersulfate, oxygen inhibits the radical polymerization and monomermixtures must be degassed prior to initiation.

The details of the preparation and the use of such gels forelectrophoresis are generally and comprehensively reviewed by B. D.Hames in B. D. Hames and D. Rickwood, Eds., "Gel Electrophoresis ofProteins", pp. 1-89, IRL Press, Washington, D. C. (1981).

pH GRADIENT GELS (IEF)

Amphoteric materials (low molecular weight ampholytes) can be added togel formulations. Following polymerization, the ampholyte materialsmigrate in the electric field according to their pI (isoelectric points)and come to rest in zones in the order of their pI. A pH gradient isthus produced in the gel.

Further details of IEF separations are described by B. An der Lan and A.Chrambach, in B. D. Hames and D. Rickwood, "Gel Electrophoresis ofProteins", pp. 157-186, IRL Press, Washington, D.C. (1981).

Note that much of the general literature describe gels of 500 μm to 1500μm thickness. However, selected works do disclose thin and ultrathingels in the range of 20 μm to 500 μm thicknesses. B. J. Radola andreferences therein, in: B. J. Radola, Ed., "Electrophoresis '79", WalterDe Gruyter, New York (1980), pp 79-94 discuss ultrathin-layerisoelectric focusing in 50-100 μm polyacrylamide gels (no gradient)prepared by casting on silanized glass or polyester sheets. B. J.Radola, A. Kinzkofer, and M. Frey in: R. C. Allen and P. Arnaud, Eds.,"Electrophoresis '81", Walter De Gruyter, New York (1981), pp 181-189describe isoelectric focusing in ultrathin-layer (20-50 μm)polyacrylamide gels. No discussion appears as to the gel preparation. A.Gorg, W. Postel, R. Westermeier, E. Gianazza, and P. G. Righetti, ibid.,pp. 259-270 also describe isoelectric focusing and gradientelectrophoresis in 240-360 μm thick poly-acrylamide gels. The gels arecast vertically, one at a time, by gradient mixing of solutions to formthe pore-size gradient. Advantages of ultrathin gels are discussed.Gradients of pore size are achieved only after great difficulty for thingels using the methods of the prior art. As a result the advantageousfeatures of thin gels have not been widely used in the prior art.

Gels prepared by any of the above processes can suffer from severaldisadvantages which can compromise their utility in polyacrylamide gelelectrophoresis (PAGE) and in isoelectric focusing (IEF). Unreactedpolymerization initiator which is present can react with biologicalmolecules and cause distorted separations or sample decomposition. Thepresent polar inorganic initiators (persulfate/TEMED) can increasebackground staining and thus decrease contrast of background with samplespots. The initiator systems described herein are advantageous in thatthey do not react or associate with biological molecules, mainly becausethey are organic in nature, not readily soluble in the case ofheterogeneous initiators. These initiators also show no problems withstaining and destaining of the gels.

In general, the electrophilic gels prepared using the various prior artmethods suffer from many disadvantages. Among these, the presence ofvarious initiators in a gel often caused random reactions of theinitiator with free monomer, buffers or acrylamide polymer. Furthermore,the inorganic initiator or its by-products may react with the proteinsamples themselves, thereby distorting the electrophoretic results.Because of the ineffective mechanical blending of reagents anduncontrolled reactions, the gels produced by the techniques of the priorart are neither accurate nor are they highly reproducible. Anotherproblem encountered with the prior art techniques is that because of thethermal convection, vibration, mixing and capillary action, it isrelatively difficult to produce thin gels, i.e., those less than 500 μmin thickness. Further, these prior art techniques tend to be relativelyexpensive since they are batch-type operations and labor-intensive.

SUMMARY OF THE INVENTION

According to the present invention, a porous gel product useful forelectrophoretic separations is obtained. This product is characterizedby the presence of a photoinitiator or a hydrogen donor and is stable,reproducible and has a controlled electrophoretic porosity.Polymerization is initiated by homogeneous photopolymerization usingwater soluble organic photoinitiators or heterogeneousphotopolymerization in which the organic initiator is suspended ordispersed in the aqueous photopolymerization solution and is controlledto produce the desired pore gradient. The gel product consistsessentially of polyacrylamide and water, but may also containacrylamide, agarose, bis-acrylamide, and other monomers or polymers.Buffers or ampholytes, detergents and solutes may also be included inthe formulations. The formulations are polymerized and cross-linked in adefined manner which may be mathematically defined for a specific use.Gel products may vary in thickness from 50 82 m to 2 mm with the 100-300μm range being most preferred.

Preferably the gel product consists of an aqueous-swelled porous matrixformed from polymerized and cross-linked acrylamide monomers. Theconcentration of acrylamide monomers in the solutions from which gelsare made is essentially from about 3% (weight/volume) to about 30%(weight/volume), and about 0.3 to 1.2% weight/volume of a water-solubleor water dispersible components of a photoinitiating system. The gelproducts may have length, width and thickness dimensions and the poresize of the gels may be in the form of a gradient, which may vary alongany of the length and width dimensions and may be in a linear, or acomplex function of pore size.

According to the method of this invention, charged bioorganic moleculesare electrophoretically separated using the gel product by the steps ofplacing a sample of bioorganic molecules on a thin plate of the gelproduct and applying a voltage across a dimension of the product. Thisgel product also has length, width, and thickness dimension and has aporosity gradient along one of the dimensions.

A process for preparing a porous gel product useful for electrophoreticseparation by the steps of, forming a water solution or dispersion of amixture from about 3% weight/volume to about 30% weight/volume ofacrylamide monomer and a cross-linking agent, the cross-linking agentcomprising about 2% weight/weight to about 15% weight/weight of thetotal monomer; adding a free radical generating system that absorbslight and initiates polymerization of the monomers; forming the solutioninto the shape of the desired gel products; and subjecting the formedsolution to light radiation to polymerize and cross-link the monomersolution.

The free radical generating system (photoinitiating system) consistsessentially of aqueous soluble or dispersible compounds that include[mono and dicarbonyl compounds, Michler's ketone[4,4'-bis(dimethylamino)benzophenone], 4-carboxybenzophenone,benzophenone, 9,10-phenanthrenequinone 3-sulfonate potassium salt,1,2-napthoquinone-2-sulfonate potassium salt,1,4-napthoquinone-2-sulfonate potassium salt,4-trichloromethyl-4-methyl-2,5-cyclohexadienone and other aromatic watersoluble or dispersible mono and dicarbonyl compounds; benzoin etherssuch as benzoin methyl either and other benzoins and dyes such asmethylene blue and new methylene blue.

The mono and dicarbonyl compounds and dyes are used with hydrogen donorsconsisting of:

N,N-dimethylaminobenzoic acid

N,N-dimethylaminoethanol

N-methyl diethanol amine

Sodium p-toluene sulfinate

Triethanolamine.

The cross-linking agent includesN,N'-(1,2-dihydroxyethylene)-bis-acrylamide,N,N'-methylene-bis-acrylamide (BIS), N,N'-diallyltartramide (DATD),ethylenediacrylate, and N,N'-bis-acrylylcystamine and othercross-linking monomers. For a given composition, the process includes astep of regulating the light intensity applied to the monomer solutionto vary porosity. Light intensity is controlled using the array oftechniques common to photographic imaging. The flux may be modulated forfixed sheet-like gels by a computer and microprocessor programmed,moving shutter. Alternatively, the light flux can be modified forcontinuous manufacture by conveying or moving individual gel molds orcontinuous or undivided gel compositions under gradient neutral densityfilters, grids, or variable shaped apertures, which modulate the lightintensity.

The gel products prepared in the manner described have many advantages,among these are enhanced electro-phoretic properties. For example, thegradient can be versitally programmed and can be accurately and reliablyreproduced by photographic imaging techniques. Furthermore, thinner gelsare readily produced using the method of this invention as well ascustom gels, i.e., gels having any pattern porosity profile desiredbecause the gradient mixing steps are avoided. In general, inexpensivegels may be produced because of the continuous process permitted by themethod of this invention. The gels are safer and the user does not haveto handle toxic acrylamide, only the packaged gel. Furthermore, the gelsmanufactured by this invention can enable the application of highervoltages with faster migration times. Additionally, the gel products ofthis invention have reduced endosmosis flow due to the reduced ioniccontent of the gel formulation.

BRIEF DESCRIPTION OF THE DRAWINGS

The detailed operation of the method described briefly above can be bestunderstood by reference to the following drawings in which:

FIG. 1 is a block diagram of the method for generating electrophoreticgels via ultra-violet or visible radiation;

FIG. 2 is a top plan view of a stepped exposure gradient slit;

FIG. 3 is a drawing illustrating the resulting gel porosity profile fromthe stepped exposure gradient slit in FIG. 2;

FIG. 4a-d is a drawing illustrating different typical slits used forporosity gradients;

FIG. 5 is a top plan view of a gel mold and film support;

FIG. 6 is a cross-sectional view in elevation taken along line 6--6 ofFIG. 5;

FIG. 7 is a bottom plan view of a gel mold and film support assembly;

FIG. 8 is a cross-sectional view in elevation taken along line 8--8 ofFIG. 7;

FIG. 9 is a drawing of a shutter system for use in the manufacturing ofelectrophoretic gels;

FIG. 10 is a drawing illustrating the top view of a shutter system foruse in the manufacturing of electrophoretic gels;

FIG. 11 is a drawing of a conveyor system for use in the manufacturingof electrophoretic gels;

FIG. 12 is a drawing illustrating a typical slit, assembly used duringgel irradiation;

FIG. 13 is a drawing illustrating the top view of the slit shown in FIG.12;

FIG. 14 is a drawing illustrating a continuous coating procedureutilizing a cover sheet; and

FIG. 15 is a drawing illustrating a continuous coating andpolymerization procedure utilizing an inert coating-radiation chamber.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT METHOD OF THE INVENTION

The method of the invention, which overcomes many of the difficultiesexperienced in the prior art, permits one to control the degree ofpolymerization of a gel to a known reproducible porosity profile. It hasbeen shown in the prior art that inorganic initiators interfere with theseparation of some biological molecules which is due to the highly polarnature of the inorganic initiator. This invention utilizes initiatorsthat are organic in nature and therefore are less polar in naturelending to a reduction of interference with said biological molecules.This invention also utilizes light radiation as a means for theinitiation of radicals in the gel solution which in turn results in gelpolymerization. The degree of polymerization and cross-linking of agiven monomer formulation in turn defines the electrophoretic gelseparation characteristics which are the key to protein separation inelectrophoretic gels. This photopolymerization technology enables thepreparation of all types of polyacrylamide electrophoresis gels. Thistechnology enables control over the porosity and electrophoreticproperties of the resulting gel materials to a degree which to date hasbeen unobtainable. Using this approach, different types ofelectrophoresis gels can be successfully developed for a broad range ofanalytical applications including pore gradient gels for molecularweight determination and pH gradient gels for resolution on the basis ofmolecular charge and isoelectric point.

Referring to FIG. 1, there is seen the steps of the method of thisinvention which may be used to produce electrophoretic gel productscharacterized by a presence of a photoinitiator which consistsessentially of an aqueous-swelled porous matrix formed from polymerizedand cross-linked monomers whereby the polymerization is initiated byradicals produced by light radiation. In FIG. 1, steps 20 through 60specifically define these essential elements which in combinationprovides a unique approach in programming the porosity of a given gelcomposition. The steps are now described.

First the gel is formulated in step 20 by selecting the gel componentswhich, depending upon the chemical compositions chosen, influence theability of the gel materials to polymerize and cross-link to yield thedesired gel porosity and influence other separation characteristics. Inorder to establish the exposure parameters for a given gel formulation,it is necessary to determine the radiation characteristics of the lightsource 30. Once this is determined, the light source is modulated 40 toproduce a gradient gel by way of a mask or mechanical shutter, whichprovides an intensity modifying means, which in turn allows for complexgel porosity patterns to be established.

This ability to control the gel porosity as a function of lightradiation produces a stable, accurate and reproducible gradient whichcan be easily adapted to a continuous manufacturing operation.Therefore, production considerations 50 are warranged. All of the abovesteps lead to the product 60 of this invention.

GEL FORMULATION 20

When considering manufacturing electrophoretic gels via light radiationin accordance with this invention, the chemical compositionconsiderations, Step 20, are important in order to achieve the desiredporosity profile. For instance, the selection of gel materialsinfluences gel characteristics such as the specific or nonspecificbinding of proteins or the electro endosmosis.

Acrylamide is the preferred and major monomer used in gel manufacturing;however, other water soluble monomers which can undergo radicalinitiated polymerization can be used. Other water soluble monomers maybe co-polymerized with acrylamide to achieve desired properties. By theprocess disclosed, certain water soluble polymers can also be used inconjunction with the above monomers or directly to form gel products.

The concentration of the above described monomers in the initial gelformulation and in the final gel products may vary from about 3% wt/v toabout 30% wt/v. This range is preferred for reasons of gel stability,strength and porous matrix properties. Gels with a low concentration ofacrylamide are the most porous and pass large molecules easily. Theseare useful in IEF where no restriction of molecules is desired or inseparation of large DNA fragments. Gels with a higher concentration ofacrylamide are less porous and provide restrictive passage for higherresolution of low molecular weight materials.

Chemical cross-linking can occur in the gels by the presence of suitablepolyunsaturated, functional acrylic or allylic compounds.

Compounds which act as suitable cross-linking agents include forexample, N,N'-methylene-bis-acrylamide (BIS), N,N'-diallyltartramide(DATD), ethylenediacrylate, N,N'-bis-acrylylcystamine,N,N'-(1,2-dihydroxyethylene)bis-acrylamide and TEOTA(polyoxyethyltrimethylolpropanetriacylate). BIS is preferred for reasonsof reactivity, compatability and solubility but other cross-linkingagents may be added to or used instead of the BIS. They are added to theinitial gel formulation in concentrations of about 0% of the totalmonomer concentration (wt/wt); to about 10% of the total monomerconcentratin (wt/wt); the actual amount being determined by the degreeof cross-linking required in the gel and may be determined empirically.The preferred range is about 2 to 7%. If these comonomers are added tothe primary acrylamide composition, the concentration of acrylamide maynot be less than about 50% of the total monomer concentration.

The total monomer concentration of the initial formulation (includingcross-linking agent) on a weight percent/volume basis is expressed as %T. The concentration of the cross-linking monomer is expressed as awt/wt percentage of the total monomer concentration and is called % C.

The gel formulations may be modified by the addition of polymericmaterials compatible with the electrophoretic separation technique. Suchcompounds include but are not limited to agarose, agar-agar, andpolyacrylamide (of varying molecular weights). The compounds may beadded to modify viscosity, porosity and gel strength. They can be addedto the formulation in concentrations ranging from about 0% wt/v to about20% wt/v, the preferred range is determined empirically on the basis ofeach individual gel product.

Water soluble polymers that may be useful individually or asco-components in gel formulation include polyacrylamide,polyvinylpyrrolidone, polyethylene oxide, polymethylvinylether,polyvinyl-alcohol and agarose.

The initial gel formulation and hence the final gel itself may befurther modified by the addition of varying concentrations of buffers,detergents, denaturants, ampholytes, and solutes. Buffer systems usedare dependent on the final end use of the gel product. Typical examplesof individual buffers, used in aqueous systems, includetris-(hydroxymethyl) aminomethane (TRIS)hydrochloric acid, citric acid,sodium hydrogen phosphate, and borates. Buffers may be used individuallyor in combination to insure proper buffering capacity and ionicstrength. The concentrations and mixtures used in such buffer systemsare selected on the basis of final end-use and are obvius to one skilledin the art. Details may be found in general references such as Hames andRickwood.

Detergents may be added to gel formulations or directly to thebioorganic sample to be separated. In both cases, the detergent is addedto solubilize the sample or to maintain a uniform charge to mass ratioso that samples separate solely on the basis of size. Detergents andtheir concentrations are selected empirically on the basis of gelproduct end-use and on the basis of sample type to be separated.Detergents which may be used include, for example, cetyltrimethylammonium bromide, cetylpyridinium chloride, deoxycholate, sodium dodecylsulfate (SDS), polyethylene oxide sorbitan monooleate and ethoxylatedoctylphenols. General references such as Hames and Rickwood treat indetail the use of such detergents.

If the gel products are to be utilized for IEF separations, ampholytesmust be added to the gel formulation. Ampholytes are amphotericelectrolytes added to IEF gels to generate the pH gradient necessary forIEF separation. The ampholytes sold commercially are generally complexmixtures of polybasic amines and polyacids. There are no limitationsimposed by the instant invention on the type or concentration ofampholytes added to the instant gel products. References such as Hamesand Rickwood clearly define the limitations of ampholytes inherent inall gel mixtures.

The initial gel formulations of the instant invention may or may not bedegassed by inert gas (nitrogen, argon, helium, etc.) purging to removedissolved oxygen prior to casting. The use of high intensity lightradiation for polymerization generates a high level of radicals, rapidlydepletes dissolved oxygen and thus obviates the usual need to rigorouslyexclude oxygen from the initial gel formulation as long as samplepreparation and exposure conditions remain constant and consistent.However, during exposure a cover sheet is necessary to preventinhibition of polymerization due to atmospheric oxygen, which can berapidly replenished by diffusion.

Once mixed, the initial gel formulation may be modified with furtheradditives, if desired, and then is placd in a casting mold or simplybetween two cover sheets. Details of the casting are described laterunder production considerations 60.

Several photoinitiators, i.e., free-radical generating systems that arecharacterized by producing gels with excellent resolving and destainingproperties, are presented in Table 1.

                  TABLE 1                                                         ______________________________________                                                Initiator System              Gel.sup.c                               Formula-                                                                              Components                    Time                                    tion No.                                                                              for 20T, 5C gels                                                                            gms.sup.a %.sup.b                                                                             (sec)                                   ______________________________________                                        1       4-carboxy-                                                                    benzophenone  0.45-0.90 0.11  25                                              triethan-                                                                     olamine       2-4       0.5-1.0                                       2       4-carboxy-                                                                    benzophenone  0.45      0.11  35-50                                           4-dimethylamino                                                               benzoic acid  2-4       0.5-1.0                                       3       4-carboxy-                                                                    benzophenone  0.45      0.11  20-90                                           N,N--dimethyl-                                                                aminoethanol  2-4       0.5-1.0                                       4       4-carboxy-                                                                    benzophenone  0.45      0.11  20-30                                           N--methyl dieth-                                                              anolamine     2-4       0.5-1.0                                       5       4-carboxy-                                                                    benzophenone  0.45      0.11  15                                              Sodium para-                                                                  toluene                                                                       sulfinate     4         1.0                                           6       9,10-phenanth-                                                                renequinone-2-                                                                sulfonate potas-                                                              sium salt     0.4       0.1   60                                      7       9,10-phenanth-                                                                renequinone-2-                                                                sulphonate potas-                                                             sium salt     0.4       0.1   25                                              triethanolamine                                                                             4.0       1.0                                           8       9,10-phenanth-                                                                renequinone-2-                                                                sulfonate potas-                                                              sium salt     0.4       0.1   25                                              sodium para-                                                                  toluene                                                                       sulfinate     4.0       1.0                                           9       9,10-phenanth-                                                                renequinone-3-                                                                sulfonate potas-                                                              sium salt     0.4       0.1   60                                              sodium paratoluene                                                                          4.0       1.0   10                                              sulfinate                                                                     1,2-napthoqui-                                                                none-4-sulfonate-                                                             potassium salt                                                                              0.4       0.1   50                                              sodium paratoluene                                                            sulfinate     4.0       1.0   11                                              1,4-naptho-                                                                   quinone-2-                                                                    sulfonate                                                                     potassium salt                                                                              0.4       0.1   40                                              sodium para-                                                                  toluene sulf-                                                                 inate                                                                 12      benzoin methyl                                                                ether         0.4       <0.1  30                                              ethanol       20        5                                             13      new methylene                                                                 blue          0.1       0.025 30.sup.d                                        sodium para-                                                                  toluene sulf-                                                                 inate         4.0       1.0                                           14      methylene blue                                                                              0.1       0.025 15.sup.d                                        sodium para-                                                                  toluene sulf-                                                                 inate         4.0       1.0                                           ______________________________________                                         Legend                                                                        .sup.a gms of component/100 ml of solution                                    .sup.b % w/volume, gel solution                                               .sup.c exposed w/2 kw mercury photopolymer source (Berkey Ascor Co.) Gel      times are for uniform gels.                                                   .sup.d gel thermally unstable; polymerized without light; not useful for      gradient gels.                                                           

It may be seen in the above table that using a two kilowatt mercuryphotopolymer light source can produce a gel at exposure time as short as15 seconds, depending upon the initiator and hydrogen donor combinationused.

RADIATIVE CHARACTERISTICS OF LIGHT SOURCE 30 AND LIGHT SOURCE MODULATION40

In order to successfully control the porosity across an electrophoreticgel product, the uniformity of the resultant radiated pattern must beknown.

This pattern must be taken into consideration in designing the slits,neutral density filters or shutter program to compensate fornonuniformity. The technique used to establish the above characteristicsare well known in the art. For example devices such as a spectralradiometer, can be used to gather exposure distribution information as afunction of a given X,Y coordinate.

In the present invention the preferred light source is ultra-violethaving an output between 300-400 nm. This correlates best with thephotoinitiators that are preferred. Actually any light source that willactivate the initiators to produce polymerization can be used. Thepreferred radiation source used was a long-wave UV lamp made byCole-Parmer Instruments consisting of two 15 watt self-filteringblack-light blue tubes which produces a 108° beam and a typicalintensity of 1100 μW/cm² of 365 nm light at 0.5" from the light housing.Another source successfully used in polymerization of gel solutions wasa 2 kw Addalux mercury photopolymer lamp with output from 300-600 nm.With the latter source the gels were located 4 inches (about 10 cm) fromthe lamp housing. The gel time for this source was 15 sec. Gel time withthe black-blue lamp is 30 sec. (2× that of the Hg photopolymer source).The second source has considerable infrared output and continuousexposure longer than 3 min would require cooling of the gel duringpolymerization.

Once the light source has been characterized, the intensity can beregulated to vary the gel porosity. The regulation is accomplished bymodulating the light flux (watts/cm²) as a function of the length, widthand thickness of the gel. The flux may be modified for a fixedsheet-like gel by a programmed shutter or a moving conveyor. Where aconveyed web or moving sheet-like gel production operation is desired,such devices as neutral density filters, grids, or variable shapedapertures, may be used to regulate the flux. In a fixed gel productionoperation, a computer driven shutter, neutral density filter or gridscan also be incorporated. More detailed discussion of these devices willbe given in the section pertaining to the generation of the exposuregradient.

EFFECTIVE POROSITY (T) AND INTENSITY (I) AS APPLIED TO GELPOLYMERIZATION VIA LIGHT RADIATION

Polyacrylamide electrophoresis gels are cross-linked polymer networksformed by polymerizing solutions normally containing 5-30% totalacrylamide monomer in water. Normally 2-7% of the monomer isN,N'-methylene-bis-acrylamide (BIS). This is added to providecross-links to the matrix. Normally the monomer is chemicallypolymerized to completion using standard persulfate initiatorcombinations, or photochemically using riboflavin. The restrictivenature of the gel is related to the percent of total monomer (% T) andthe amount of cross-linker present via polymer density and degree ofcross-linking.

Literature convention has defined the restrictive nature of a gel interms of the percent total monomer containing 3% BIS polymerized in thegel. For example, a gel having a "20% T porosity" demonstrates therestrictive character of a gel formed by polymerizing to completion asolution of 20% total acrylamide to which 3% BIS had been added.Therefore, one refers to gels as having 10, 15, 20% T porosity dependingon the composition.

This above convention works well in relating the electrophoreticresponse of the gel formulation since polymerization is assumed toapproach completion. In the context of the present invention, theelectrophoretic response cannot be related directly to the acrylamidecontent or % T. This is because both degree and nature of thepolymerization process is controlled by the radiation exposure. Forexample, using photoinitiated radiation polymerization, a range ofporosity can be achieved with a given initial formulation by varying therange of exposure. The equivalent porosity of a photoinitiated producedgel can be referred to as an equivalent effective % T, which will betermed T in the content of this disclosure.

GENERATION OF EXPOSURE GRADIENTS

The most common electrophoretic gel performance relationships used inthe art is molecular weight versus distance and porosity versus distanceprofiles. The accuracy by which the porosity can be controlled as afunction of distance across a gel is unique allowing for essentially aprogrammable profile which is dependent upon desired performancerelationships. In accordance with this invention, of porosity iscontrolled via photoinitiation and a gel can be manufactured to thespecific needs of a particular researcher.

The generation of the porosity gradient incorporates a radiation fluxmodifying means for both fixed and conveyed or moving gels. This fluxmodifying means takes the form of moving shutters, screens, grids, orvariable shaped apertures. A typical slit used for porosity gradientproduction is illustrated in FIG. 2, where depending upon the shape ofthe slit a given porosity profile is achieved. For example, using astepped exposure shaped slit as a mask 108 positioned above a gelcarried on a conveyor moving under a light source, the light sourcewould polymerize the gel 112 only in the regions of the mask which areopen to the gel. If the gel were moved or conveyed relative to the maskand light source, certain regions of the gel would be exposed for alonger length of time, i.e., more exposure. Since exposure isproportional to polymerization and cross-linking, which in turn isproportional to porosity, a pore gradient gel could be produced.

For example, in FIGS. 2 and 3, an electrophoretic gel 112 exposed tolight using the stepped slit 110 would have porosity gradients in stepfashion across the lanes 116 through 126. Lane 116 would have thehighest degree of polymerization corresponding to a smaller pore size128, since the vertical slit length 130 of lane 116 is the largest ofthe mask. Lane 126 would correspond to the least polymerized portion ofthe gel corresponding to a larger pore size 114. Other examples ofdifferent exposure gradient masks are illustrated in FIG. 4a, b,c,d,such as a linear 130, polynomial 132, exponential 134, or stepped dosepatterns 136.

For fixed or stationary gel exposure, porosity gradients can be createdby the use of a programmable shutter assembly which moves across the gelat a prescribed speed. The speed of the shutter as it moves across a gelfor a given intensity, and gel composition will determine the porosityprofile. A detailed description of the shutter exposure system alongwith a continuous gel manufacturing process will be discussed in thesection describing production considerations 60.

PRODUCTION CONSIDERATION 50

With the system optimized in accordance with the discussion in theprevious disclosure, it is entirely feasible to produce gels of a givenporosity profile using the batch mode or continuous manufacturingoperation.

According to this invention the gel formulations are exposed in two partmolds having a polystyrene tray 112 and having a polyester cover sheet104 which serves as a gel support, as best seen in FIGS. 5, 6, 7 and 8.The trays 112 used in a successful experiment were essentially shallow4.5"×5" trays, 12 mils (300 μm) deep and contain a series of ridgesrunning about 3/4 of the length of the gel dividing it into eight lanes.The trays 112 are covered with 7 mil "Gel Fix"® polyester film 104obtained from Serva Feinbiochemica to provide a flexible support for thecompleted electrophoresis gel. For special 2-D integrated gelapplication, electrodes are printed on the support film. In this casethe lane ridges and sample application wells are removed from the molds.

The polyester film 104 may be sealed to the polystyrene tray 112 byapplying a thin coating of Dow Corning silicone high vacuum grease tothe edge of the mold and then rolling the two parts together. The greaseprovides a water-tight liquid seal. The two parts are then clampedtogether using strips of a plastic edge moldilng 114 for paper bundles(Slide-Lock Binding Bars®) that are cut into strips the length of thegel. Following exposure, the gel adheres preferentially to the polyesterfilm 104 support and thus can be pealed out of the mold due to thedifference in adhesion between the two halves.

Prior to attachment to the tray 112, the polyester film 104 is squeezedonto the plate 102 using water to provide adhesion. The laminate is thenattached to the tray 112 using silicon grease and edge clamps asdescribed earlier. The trays 112 are filled through small ports 106located at opposite corners of the tray 112. The trays 112 are held onan angle to allow air or argon to be displaced by the monomer solution.The ports 106 are capped with small rubber septa 108 and an aluminumplate 102 is pressed into place below film 104 to act as a gel support.

The mold assembly consisting of the gel formulation 110, polyester film104, tray 112, plate 102 and edge molding 114 such that plate 102 restsdirectly on the conveyor belt. This device is termed the gel supportassembly 144 and is used for gel polymerization regardless of the typeof manufacturing process chosen.

Illustrated in FIGS. 9 and 10 is a computer driven shutter device whichis capable of producing exposure gradients in a batch type mode. Theprogrammable shutter 142 is placed at that distance below the lightsource 140, which results in a uniform radiation distribution at thetarget. The gel support assemblies 144, filled with monomer asdescribed, are then placed in the sample holders 146 such that the fillports 106 are on the top side of the support assembly 144 2 or 3 abreast(as best seen in FIG. 9.) The programmable shutter 142 is thenpositioned directly above the support assemblies 144. The light source140 is then activated and allowed to reach full intensity. Theprogrammable shutter 142 is then activated which starts to open via ascrew drive 148 and stepper motor 150 arrangement controlled by thesoftware commands of the computer 156 according to the desired exposureprofile. An Intel® 8255A Programmable Peripheral interface 154 was usedto coordinate the analog to digital control communications between thecomputer and shutter drive system. To isolate the user from theultra-violet radiation, a radiation cell wall 152 is located between theshutter system and control room. A top view of this apparatus isillustrated in FIG. 10. After exposure is complete the light source issimultaneously cutoff.

A second device used for gel formulation exposure is a conveyor systemfor continuous manufacturing of gels, which is illustrated in FIG. 11.The gel support assemblies 144 are filled with monomer by hand and arethen placed on a stainless steel conveyor belt 160, which is used totransport the gels to the light source 164 and then on to the collectionarea 180.

After placing the gel support assemblies 144 on the conveyor belt 160,the light source 164 is activated. When the light has stabilized, theconveyor 160 is started and the gel support assemblies 144 pass under aslit, grid or screen assembly 168 at a constant predetermined beltspeed.

An illustration of a typical slit, grid, or screen assembly 168 is shownin FIGS. 12 and 13. The slit mask 196 of interest is chosen dependingupon the desired porosity profile required. The slit 196 is coupled tothe holder and shield assembly 178, which acts as a retaining means forthe light filter of interest along with protecting the gel supportassemblies 144 from unwanted light. To allow the passage of the lightthroughout the slit 196 a window 198 is provided above the slit. Theslit 196 is held parallel to the shield assembly 198 via four bushings194.

The conveyor belt 160 (FIG. 11) is driven by a motor 170, which iscoupled to a reduction gear box 172, which drives a belt 174, which inturn drives a belt pulley 176. During exposure the gel supportassemblies 144 are protected from unwanted light by the shielding 178,located above the conveyor 160 and sample collection area 180. As thegel support assemblies 144 leave the conveyor 160 they slide down anexit ramp 182 into the sample storage area 180, which can be adjustedvertically by a hydraulic stand 184.

Since the light source produces a constant intensity with constantenergy over a defined area at a fixed rate, polymerization isproportional to the time of exposure. Time can be modulated by theprogrammed shutter movement, or by constant conveyor movement undervariable fixed slits cut to dimensions corresponding to the desiredgradient.

The calculations of slit dimensions, shutter movement, conveyorbeltspeed, and light intensity are all factors which must be determinedprior to manufacturing electrophoretic gels using photopolymerization.

Modifications can be made to the conveyor system to provide continuousintroduction and removal of samples which are compatible with potentialmanufacturing processes.

An example of how each of the above manufacturing systems can be used togenerate uniform porosity exposures, separate lane porosity variationexposures, and continuous gradient exposures is described below.

UNIFORM EXPOSURES

To produce a uniform exposure using the batch type shutter system, seeFIG. 9, the samples 144 are placed on the shutter table 146 so that eachportion of the gel 144 is exposed for the same length of time. In theconveyor system FIG. 11, the samples 144 are passed under a square orrectangular slit 196 at constant rate. These two manufacturing methodsare subtly different from each other even though they both produce thesame overall exposure. With the shutter system, the sample 144 receivesessentially a uniform instantaneous exposure over its entire surfacearea simultaneously initiating polymerization at all points in the gel.With the conveyor system an exposure front passes over the gel producinga corresponding polymerizatin or reaction front, along with itsassociated diffusion and thermal gradients.

LANE EXPOSURES

To produce a gel with separate lanes having different but uniformexposures using a batch type system, see FIGS. 9 and 10, requires themanual or automatic movement of the shutter 142 across the length of thegel in a stepped fashion to mask the sample in an exposure patternsimilar to the one described in FIGS. 2 and 2. The preferred method ofproducing "lane" exposures is the use of the conveyor system, FIG. 11,with a slit pattern the same as that described in FIG. 2.

CONTINUOUS GRADIENT EXPOSURES

The final kind of exposure used was one to produce exposure gradientscorresponding to a specific desired porosity range or limiting molecularweight distribution along the length of the gel in the electrophoresisdirection. These gradients were produced using both shutter and variablefixed slits with the conveyor. The use of each will be described.

Using the batach type shutter system, FIGS. 9 and 10, the supportassemblies 144 are placed in the sample holders 146. The programmableshutter 142 is then positioned direclty above support assemblies 144.The light source 140 is then activated and allowed to reach fullintensity at which time the shutter 142 is opened to produce the desiredexposure profile.

The preferred means of exposure is using the conveyor system (FIG. 11),with the use of masks 168 to produce the gradient. The masks 168 are cutin specific patterns required to produce the exposure gradients neededto generate particular molecular weight or porosity versus distanceprofiles. Sample gels 144 are placed on the conveyor with theelectrophoresis direction perpendicular to the direction of the beltmovement and then exposed. FIG. 4a-d illustrates differentrepresentative mask patterns available to give desired electrophoreticporosity responses.

COATING PROCEDURE

While a casting procedure using molds must be used for making gels frompure water monomer formulations, a continuous coating procedure can beused with solutions that have been thickened using either polyacrylamideor some other compatible, water soluble polymers. Two possible coatingtechniques will be described. The first utilizes a cover sheet, thesecond an inert coating-reaction chamber.

If the gel formulations are coated at a remote location from the lightsource, a cover sheet 210 as best seen in FIG. 14, will have to beapplied to prevent evaporation of solvent (water) and provide an oxygenbarrier for polymerization. The cover sheet 210 may also contain printedelectrodes and the like, or these may be included as part of the base.To provide uniform gel thickness, spacers 212 must be used. These areplaced on the film support 214 as a template. The monomer-polymercomposition 218 is then pumped onto the film support 214 by way of anozzle 220. A coating knife 216 is used to form the monomer-polymerformulation 218 into a uniform gel.

If the monomer-polymer composition has sufficient physical integrity, acoating of predetermined thickness can be made using a doctor knife 222,which is housed in a U-shaped bracket which provides support during gelmanufacturing as best seen in FIG. 15. As in FIG. 14, themonomer-polymer composition 218 is pumped by way of a nozzle 220 ontothe film support 214. The gel is then formed via the doctor knife 222and then polymerized by the light source 228, in an inert atmosphere226. Temperature and humidity must be regulated to prevent solvent lossduring coating and irradiation. Both of the methods described above canbe used for irradiating thickened monomer solutions or pure watersoluble polymer solutions to produce a given gel product 224.

Typical photopolymerization SDS-PAGE formulation for gradient anduniform gels are set forth in Example 1, and for IEF gels in Example 2.

The following example illustrates the preparation of SDS-PAGEelectrophoresis gel formulation for preparation of photopolymerizationgels using a homogeneous initiating system. The free-radical generatingsystem which includes 4-carboxybenzophenone and the hydrogen donortriethanolamine is particularly preferred.

EXAMPLE 1

Stock solutions were prepared as follows:

MONOMER SOLUTION

Acrylamide: 80 g

N,N'-methylene-bis-acrylamide: 2.1 g

Distilled water to make 200 ml

BUFFER SOLUTION

Tris(hydroxymethyl)amino methane: 18.1 g

Sodium dodecyl sulfate: 0.4 g

Distilled water: 80 ml

pH adjusted to 8.8 w/HC1:

Distilled water to make 100 ml

INITIATOR SOLUTION

4-carboxybenzophenone: 0.90 g

Triethanolamine: 2.00 g

Distilled water: 85 ml

pH adjusted to 8.8 w/HC1 or NaOH

Distilled water to make 100 ml

GEL SOLUTION

Gel solutions were prepared by mixng the stock solution as follows,

50% monomer solution

25% buffer solution

25% initiator solution

The gel solution was introduced into shallow trays, such as thosedescribed above, 4.5"×5" and 12 mils deep. After filling the trays werecovered with a polyester film as described above.

SDS-PAGE electrophoresis gels were prepared as indicated above using anexposure time of 60 seconds, and were evaluated for both uniform andgradient exposed samples. Shorter exposure times can be used, butoptimum resolution occurred at about 60 seconds using the black lightsource noted above. These samples were evaluated using a BLR standardmixture of proteins consisting of:

Insulin

Bovin Try, Inhibitor

Cytochrome C

Lysozyme

β-lactoglobulin

α-Chymotrypsinogen

Ovalbumin.

The initiating system so stated yielded gels having good resolving anddestaining characteristics. The above protein mixture was resolved intosix components by uniform exposure and into seven components forgradient exposed gels; longer exposure times were used for gradient gelsto compensate for a neutral density filter when used; in additiongradient exposed gels showed sharper bands. Reproducibility wasdemonstrated using the formulations so stated.

The following example illustrates the preparation of IEF gelformulations for preparation of photopolymerization gels.

EXAMPLE 2

The following examples illustrate applicability of the compositions ofthis invention for production gels for isoelectric focusing. Stocksolutions were prepared as follows:

STOCK MONOMER SOLUTION

Acrylamide: 6.47 gms

Bis-acrylamide: 0.20 gms

Deionized water to make: 66.7 ml

AMPHOLYTE STOCK SOLUTION

Pharmalyte® (pH 3 to 10): 2.5 ml

Glycerol: 4.0 ml

Deionized water to make: 10 ml

INITIATOR STOCK SOLUTIONS

Benzoin methyl ether: 0.4 gms

Ethanol: 10 ml

Deionized water to make: 50 ml

Adjust to pH 7.0.

GEL SOLUTION

Gel solutions were prepared by mixing stock solutions as follows:

50% Monomer solution

25% Ampholyte solution

25% Initiator solution.

Gels were prepared as in Example 1. However, exposure times were longerthan with SDS-PAGE gels which contain charged initiating species with aminimum of 10 minutes exposure necessary to produce a functional gel. Tobe assured that polymerization was complete, exposures of 20 minutesusing the black light source noted above were made. The longerpolymerization however is not due to inefficiency of the initiator butdue to the lower acrylamide monomer concentration required for IEF gels(generally 5-7 T/2-5%C vs. 7-30T/2-7% for SDS-PAGE Gels). IEF gelsrequire use of neutral initiating components to avoid interference withpH gradient. Gels with good resolution (a pharmacia standard broad PIcalibration kit used) and destaining properties were obtained fromsolutions prepared as indicated in Example 2. Incorporation of benzoinmethyl ether (a neutral initiator that does not require use of ahydrogen donor) was accomplished by addition of ethanol as a co-solvent(5% in gel solution). Ethanol doesn't appear to have adverse effects onelectrophoresis.

GEL PRODUCTS 60

Using this invention to induce electrophoretic gel polymerization viaphotoinitiation it is possible to electrophoretically separatebioorganic molecules using the gel products 60 set forth in the abovedisclosure by the steps of placing a sample of bioorganic molecules on athin plate of the gel product and applying a voltage across a dimensionother than the thin dimension of the plate. The gel products may vary inthickness from 50 μm to 2 mm with the 100-300 μm range being mostpreferred. Also, depending upon the desired electrophoretic separationrequired, the gels may be designed for specific molecular weight orporosity versus distance distributions across a gel or have uniform,lane, or gradient porosity profiles. The advantages of suchphotopolymerized gels produced in accordance with this invention aresummarized as follows:

ACCURATE AND REPRODUCIBLE GELS

The precise control of sample formulation and preparation, lightexposure and sample placement affords excellent reproducibility in gelpreparation. This translates into high reproducibility of gradient shapeand type.

CUSTOM GELS

Gel gradients may be customized to any pattern porosity profile throughmicroprocessor control of light shutter, slit or mask. For example,discontinuous gradients and gradients of complex profiles can be readilyproduced.

CONTINUOUS PRODUCITON OF GELS

Currently, gels are batch prepared. The instant case allows continuousproduction. Each gel can thus be made rapidly under reproducibleconditions.

THINNER GELS

The photopolymerization process enables the production of thin gradientgels (500μ) which are not readily produced by conventional processes.Since thin gels require less power to run they can be electrophoresedfaster and are easier to handle. This is faster and saves energy.

SAFER GELS

Since the user does not have to handle toxic acrylamide, the packagedgel materials can be more safely handled.

STABLE GRADIENT

Unreacted initiators in other systems may cause polymerization of freemonomer or cross-linking of polymer to occur after gel preparation iscomplete. Thus cross-link density and gradient can changeuncontrollably. The initiators described herein do not causepolymerization to occur in the absence of light and thus provide astable electrophoretic gel system.

What is claimed is:
 1. A process for preparing a porous gel product,defining a volume, useful for electrophoretic separation the gel productcharacterized by the presence(d) of a photoinitiator, having a constantatomic composition over its volume, and length and width dimensions, andwhich is stable, reproducible and has a controlled porosity gradientalong one of the length and width dimensions by the steps of:forming awater solution or dispersion of a mixture of from about 3% weight/volumeto about 30% weight/volume of acrylamide monomer and a cross-linkingagent, the cross-linking agent comprising about 2% weight/weight toabout 15% weight/weight of the total monomer, the crosslinking agentconsists essentially of N,N'-(1,2-dihydroxyethylene)-bis-acrylamide,N,N'-methylene-bis-acrylamide (BIS), N,N'-diallyltartramide (DATD),ethylenediacrylate, N,N'-bis-, acrylylcystamine, andpolyoxytehyltrimethylolpropane triacrylate (TEOTA) adding a free radicalgenerating system that absorbs ultraviolet light radiation and initiatespolymerization of the monomers, the free radical generating systemconsists essentially of aqueous soluble or dispersible compounds thatinclude 4-carboxybenzophenone, benzophenone, 9,10-phenanthrene-quinone3-sulfonate potassium salt, 1,2-napthoquinone-2-sulfonate potassiumsalt, 1,4-napthoquinone-2-sulfonate potassium salt,4-trichloromethyl-4-methyl-2,5-cyclohexadienone and other aromatic watersoluble or dispersible mono and dicarbonyl compounds, benzoin etherssuch as benzoin methyl ether and other benzoins, forming the solutioninto the shape of the desired gel product, subjecting the formedsolution to ultraviolet radiation to polymerize and cross-link themonomer solution or dispersion, and modulating the exposure of theformed solution to the ultraviolet radiation over the length and/orwidth of desired gel product according the desired porosity gradient. 2.The process set forth in claim 1 wherein the mono and dicarbonylcompounds and dyes are used with hydrogen donors consisting essentiallyof:N,N-dimethylaminobenzoic acid, N,N-dimethylamino ethanol, N-methyldiethanolamine, Sodium p-toluene sulfinate, and Triethanolamine.
 3. Theprocess set forth in claim 1 wherein the ampholyte is added to thesolution or dispersion and the free radical system contains benzoinmethyl ether <0.1% weight/volume.
 4. The process set forth in claim 1wherein the solution is formed into the shape of the gel product bycoating the solution onto a substrate.